1. Field of the Invention
The present invention relates to an immersion microscope objective, and more specifically to an immersion microscope objective used in multiphoton excitation.
2. Description of the Related Art
Recently, a fluorescent observation method using multiphoton excitation has received widespread attention as means for performing a fluorescent observation by a microscope. The multiphoton excitation is a phenomenon equivalent to the excitation caused by the intrinsic absorption wavelength by simultaneously irradiating phosphors with the light having a wavelength of a substantially integral multiple of an absorption wavelength.
A multiphoton excitation phenomenon is a nonlinear phenomenon and caused, for example, at a probability proportional to the square of the intensity of the excitation light in case of two-photon excitation. On the other hand, when the excitation light is condensed by an objective of a microscope, the optical density of the excitation light becomes lower at the inverse square of the distance from the focal plane. Therefore, the multiphoton excitation phenomenon occurs only in the vicinity of the focal point, and fluorescence is irradiated only from the portion. By the property, a multiphoton excitation microscope does not require a confocal pinhole used in a normal confocal microscope. In addition, since an excitation phenomenon occurs only on the focal plane, there is little fading of fluorescence in a sample.
The excitation light used in the multiphoton excitation is generally infrared light having a wavelength longer than the wavelength of normally used visible light. Generally, the longer a wavelength, the harder the light scatters (Rayleigh scattering). Therefore, although a scattering sample such as a living body sample etc. is to be observed, the excitation light can reach the deep part of the sample by the excitation by infrared light. Accordingly, the deep part of a living body which has not been observed by visible light etc. can be observed by the multiphoton excitation. In addition, since the infrared light is less phototoxic than ultraviolet light or visible light, the damage to a living body sample can be successfully suppressed.
As described above, the fluorescent observation method using the multiphoton excitation has a number of merits, it is a very effective fluorescent observation method.
On the other hand, in the fluorescent observation method using the multiphoton excitation above, the following technological demand is imposed on an objective.
First, the objective is to have a large numerical aperture, and to be appropriately aberration-corrected. To generate the multiphoton excitation, a plurality of photons are to simultaneously collide against one phosphor. To attain this, it is necessary to realize very high photon density at the focal position of the objective. Therefore, the objective is to have a large numerical aperture, and to be appropriately aberration-corrected. To be more practical, since the excitation light is infrared light, it is necessary that the aberration of the infrared light is corrected.
Second, it is necessary that the objective has a long working distance. Since a patch clamp method is often used in the multiphoton excitation microscope, it is necessary to reserve a work space between the tip of the objective and the sample. In addition, to observe the deep part of a sample, it is necessary to reserve the distance from the tip of the objective to the object surface longer than the depth of the sample when the object surface matches the focal point of the objective. Therefore, the objective is to have a long working distance.
For example, Japanese Laid-open Patent Publication No. 2005-189732 discloses an objective having a long working distance. Furthermore, Japanese Laid-open Patent Publication No. 2003-15046 discloses an objective having a large numerical aperture and a correction ring.